Complete Gram Stain Lab PDF

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Microbiology Lab Notebook Report

Gram Stain - Exercise 3-5 February 6, 2020

Introduction The Gram stain is the most important and widely used differential stain which is used to differentiate between Gram-positive and Gram-negative cells. The Gram reaction helps with identification of an organism by presumptive determination of cell morphology, size and arrangement and is typically the initial test performed in the lab.

Purpose The purpose of this lab is to differentiate with color which cells are going to have a Grampositive cell wall and which cells will have a Gram-negative cell wall. With this lab we are able to practice our smearing and heat fixing technique from the simple stain lab. Depending on how accurate the color reading is after we rinse the stain, it will be a good indicator on if performed the lab adequately.

Materials • • • •

• • • • • •

Microscope Clean glass microscope slides Sterile toothpick Gram stain solutions: - Gram crystal violet - Gram iodine - 95 % ethanol - Gram safranin Squirt bottle with water Bibulous paper Staining tray Staining screen Slide holder Recommended organisms: Pseudomonas aeruginosa & Escherichia coli

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Procedure 1. Begin lab with 3 heat fixed emulsion slides resting on the staining tray. 2. Cover the SMEARS ONLY, not the whole slide with the crystal violet stain for one minute, make sure any excess goes into the staining tray. 3. Grab the slide with a slide holder and put it at an angle. Rinse slide with distilled water into the staining tray and make sure all of the crystal violet comes off. 4. Cover the smears with the Gram iodine stain for 1 minute, make sure any excess goes into the staining tray. 5. Grab the slide with a slide holder and put it at an angle. Rinse slide with distilled water into the staining tray. 6. Holding the slide at an angle decolorize it with the Gram decolorizer by allowing it to run down the slide until the runoff is clear. 7. When run off is clear IMMEDIATELY rinse the slide with distilled water into the staining tray. 8. Counterstain the smear with the safranin for 1 minute. 9. Grab the slide with the slide holder and hold it at an angle while gently rinsing with distilled water. 10. When finished gently blot the smear dry with a tablet of bibulous paper, DO NOT RUB. 11. When dry, observe slide under the oil immersion.

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Results

Observations and Interpretations Organism or Source

Cellular Morphology and Arrangement

Color and Gram Reactions

Organism #1: Staphylococcus aureus

Cocci shaped, arranged in grape like clusters

Purple, Gram-positive

Organism #2: Escherichia coli

Bacilli shaped, with no particular arrangement

Pink, Gram-negative

Organism #3: Pseudomonas aeruginosa

Bacilli shaped, arranged in single cell structures

Pink, Gram-negative

Organism #4: Bacillus subtitles

Bacilli shaped, arranged in pairs or chains

Purple, Gram-positive

Conclusion The slides are difficult to see in the picture meaning there may not have been enough bacteria, causing very little left on the slide. This could be a cause of improper heat-fixing. On the first image, staphylococcus aureus is supposed to appear purple and escherichia coli is almost not visible. This slide was decolorized for too long.

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Follow-Up Questions 1. Predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when Performing a Gram stain. Consider each mistake independently. a. Failure to add the iodine. - Failure to add iodine in a gram stain would cause the gram-positive cell to lose it’s purple color during decolorization because the iodine is added as a mordant to enhance and solidify the crystal violet. b. Failure to apply the decolorizer. - Failure to apply the decolorizer would cause the gram-negative cells to appear grampositive and purple because the crystal violet would not be removed prior to being counter stained. Both gram-positive and gram-negative would appear the same. c. Failure to apply the safranin. - Failure to apply the safranin would result in almost transparent gram-negative cells. Safranin is the counter stain which makes the decolorized cells appear too faint and mistakenly identified. d. Reversal of crystal violet and safranin stains. - Reversal of the crystal violet and safranin stains would cause everything to appear purple at the end. During decolorization, the Safranin would be leeched out, the Mordant; Iodine, will only cross-link with the Crystal Violet. 3. If you saw large, eukaryotic cells in the preparation made from your gum line, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative? (You can make a good guess about this even if you didn't see your cells.) - No, only Bacteria are Gram-positive or Gram-negative. 4. One of your lab partners has followed the recommended procedure of running Grampositive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why? - I would suggest that she make sure she is using new cultures, because if she is using old cultures then they lose their ability to retain the Crystal Violet

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